The long-term objective is to determine the mechanism of milk-lipid secretion from mammary epithelial cells during lactation. Despite the importance of breast milk for the survival and nutrition of the suckling neonate, molecular and cellular mechanisms underlying secretion of the principal components of milk including lipid and protein remain unknown. Lactation fails in about 5% of breast-feeding mothers for unknown reasons. In this proposed work, the potential role of the milk proteins, butyrophilin (Btnlat) and xanthine dehydrogenase/ oxidase (Xdh), in the secretion of milk-lipid droplets will be studied. Btn1a1 and Xdh are the first proteins to be genetically linked to milk-lipid secretion because ablation of the respective genes severely disrupts the regulated secretion of milk lipid. We will test the hypothesis that Btn1a1 is transported to the apical plasma membrane and binds to Xdh on the surface of intracellular lipid droplets forming a linker protein complex essential for the budding and release of lipid from the cell. Interactive domain(s) in the cytoplasmic tail of Btn1a1 and in Xdh will be identified by assaying for binding between truncated forms of the proteins by GST pull-down assays and column chromatography. Interactive domains of Btn1a1 and Xdh, identified in vitro, will be screened for function in vivo by expressing wild-type or truncated functional forms of either protein from transgenes driven by a mammary-specific promoter. Wild-type Btn1a1 or its functional domain should restore the wild-type phenotype in Btn1aT'~ mice but the functional domain of Xdh should inhibit lipid secretion in wild-type mice by acting in a dominant negative manner. The intracellular distribution and targeting dynamics of Btn1a1 and Xdh will be determined by confocal microscopy of mammaryexplants prepared from glands transduced with adenoviral vectors encoding fluorescently labeled fusion proteins of either protein. The working hypothesis will be supported if Btn1a1 is transported to the apical membrane and becomes less mobile in the lipid bilayer as it is incorporated into budding lipid droplets. Xdh should bind to lipid droplets in the cytoplasm and colocalize with Btn1a1 at the apical surface. Lay language: Lactation fails in about 5% of breast-feeding mothers. This project will test the hypothesis that the milk proteins, butyrophilin and xanthine oxidase, are essential for the secretion of milk lipid and thus enhance our understanding of, and potential ability to treat lactation deficiencies in humans.